DESCRIPTION: The essential cofactor of the enzyme ribonucleotide reductase consists of an oxo-bridged diiron(III) cluster and an adjacent tyrosyl radical. The activities of existing anticancer and antiviral drugs (e.g. hydroxyurea and thiosemicarbazones) derive from their reductive disassembly of this cofactor and consequent inhibition of DNA synthesis, suggesting that an understanding of the reaction by which the cofactor is generated might be of value in design of new pharmacology. The cofactor assembles spontaneously in vitro when the apo form (lacking iron and radical) of the enzyme's R2 subunit is incubated with ferrous ions and O2. The assembly reaction comprises binding of Fe(II) by apo R2, reductive activation of dioxygen by the resulting diiron(II) cluster, transfer of an "extra electron" from a third Fe(II) or another reductant to the assembling cofactor, and one-electron oxidation of a specific tyrosine residue by an intermediate species. Previous investigations of cofactor assembly into E. coli R2 suggested 1) that formation of the oxygen-reactive Fe(II)-R2 complex is a multistep process in which a protein conformational change is rate limiting, and 2) that the protein facilitates rapid transfer of the "extra electron" to the reacting iron cluster prior to or during formation of the first observable intermediate species. The proposed research will use kinetic and spectroscopic methods in combination with protein engineering to: 1) characterize Fe(II) binding by the R2 proteins from E. coli, mouse, and herpes virus, defining the multiple Fe(II)-R2 complexes that are early intermediates in the assembly reaction and the kinetic pathways by which they form and decay; 2) define the mechanisms for the delivery of the "extra electron" and test the hypothesis that, by facilitating this step, the R2 protein ensures the observed one-electron oxidation chemistry to the exclusion of possible two-electron alternatives (as occur in related diiron proteins and in the F208Y mutant of E. coli R2); and 3) determine the chemical mechanism of the altered assembly reaction that occurs in the site-directed mutant, R2-F208Y, in which the engineered tyrosine residue 208 is ortho hydroxylated (a two-electron reaction). By providing insight into how the E. coli R3 protein directs the outcome of the assembly reaction and a foundation for characterizing cofactor assembly into the mammalian and viral R2s, the proposed research will enhance our understanding of the biogenesis of this important drug target.